1. Field of the Invention
The present invention relates to immunoassay methods for the determination of vitamin B-6, which utilize monoclonal antibodies therefor.
2. Brief Description of the Prior Art
The biological activity of the vitamin B.sub.6 group is displayed by pyridoxine, pyridoxal, pyridoxamine and their 5-phosphate esters. The biologically active coenzyme form is pyridoxal 5-phosphate, and the other compounds undergo enzymatic conversion in tissues to pyridoxal 5-phosphate. The vitamin is widely and uniformly distributed in all foods, muscle meats, liver, vegetables, and whole grain cereals among the best sources. (See Harrison's "Principles of Internal Medicine", Vol. 1, pages 427-428.)
Pyridoxal phosphate acts as a cofactor for an exceptionally large number of enzymes involved in amino acid metabolism, including transaminases, synthetases, and hydroxylases. It is of particular importance in humans in the metabolism of tryptophan, glycine, serine, glutamate, and the sulfur-containing amino acids. Pyridoxal phosphate is also required for the synthesis of the heme precursor .delta.-amino levulinic acid. A large share of body pyridoxine is found in muscle phosphorylase, where it may function not catalytically but to stabilize the enzyme. It also plays a vital role in neuronal excitability, possibly as a result of its function in transulfuration reactions or .gamma.-amino butyric acid metabolism.
The widespread occurrence of the vitamin in food is probably the reason that a naturally occurring pure pyridoxine deficiency has never been recognized except when the pyridoxine content of food is destroyed during processing, as has occurred in some processed infant formulas. However, present pyridoxine deficiency is frequent in the United States. This happens because many commonly used drugs act as a pyridoxine antagonists. Such drugs include hydrazines such as isoniazid, cycloserine, an antituberculous drug, and penicillamine. Abnormal tryptophan metabolism and convulsions brought about by these antagonists can be prevented by supplementation with the vitamin.
There are also a large number of genetic conditions in which abnormalities in vitamin B.sub.6 metabolism occur. One group, if not provided with daily supplements of pyridoxine during infancy, develops convulsions and brain damage and dies; these children have an apoenzyme for glutamic acid decarboxylase that has a decreased binding affinity for pyridoxal phosphate. Another group has pyridoxine responsive chronic anemia. (See also Mudd, S. H., "Pyridoxine-Responsive Genetic Disease" Fed. Proc. 30: 970 (1971)).
Estimates of vitamin deficiency have been based upon the cure of clinical signs of deficiency, the excretion of tryptophan metabolites after tryptophan-loading tests, measurement of various amino acid transferase activities in blood, and excretion of pyridoxine or its metabolites, or of oxalate in the urine. The most common index is the measurement of tryptophan metabolites, following tryptophan loading. Alternatively, cystathionine can be assayed after a methionine load. It is also possible to carry out measurement of red blood cell glutamic pyruvic transaminase in vitro, in the presence and absence of pyridoxal phosphate.
Other enzymic assays for pyridoxal phosphate include the pyridoxal dependence of enzymes such as tyrosine decarboxylase or tryptophanase, the activities of which vary directly with pyridoxal phosphate concentrations. Chabner B., and Livingston, D. (Analytical Biochemistry 34:413-423 (1970)) describe a method of measuring pyridoxal phosphate based on the measurement of .sup.14 CO.sub.2 evolved during the decarboxylation of L-tyrosine-1-C.sup.14 by pyridoxal phosphate dependent tyrosine apodecarboxylase from S. faecalis. This method includes a partially purified apoenzyme, a rapid assay procedure involving the apoenzyme and quantitative capture of evolved radioactive CO.sub.2 in a removable potassium hydroxide-containing well which could be placed in scintillation fluid for immediate counting.
Given the importance of pyridoxal phosphate and related metabolites, and the necessity of accurately ascertaining levels thereof in animals, it would be useful if an alternative, rapid and efficient method for its assay could be provided.
A need therefore continues to exist for an assay of vitamin B.sub.6 metabolites in animals.